Bottom Line:
Cytokine-treated islets expressing IRS2(5A) secreted significantly more insulin in response to glucose than did islets expressing IRS-2(WT).Elimination of a physiological negative feedback control mechanism along the insulin-signaling pathway that involves Ser/Thr phosphorylation of IRS-2 affords protection against the adverse effects of proinflammatory cytokines and improves beta-cell function under stress.Genetic approaches that promote IRS2(5A) expression in pancreatic beta-cells, therefore, could be considered a rational treatment against beta-cell failure after islet transplantation.

Objective: Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-1 signaling. Here, we examined the role of Ser phosphorylation of IRS-2 in mediating the inhibitory effects of proinflammatory cytokines and cellular stress on beta-cell function.

Research design and methods: Five potential inhibitory Ser sites located proximally to the P-Tyr binding domain of IRS-2 were mutated to Ala. These IRS-2 mutants, denoted IRS-2(5A), and their wild-type controls (IRS-2(WT)) were introduced into adenoviral constructs that were infected into Min6 cells or into cultured murine islets.

Results: When expressed in cultured mouse islets, IRS-2(5A) was better than IRS-2(WT) in protecting beta-cells from apoptosis induced by a combination of IL-1beta, IFN-gamma, TNF-alpha, and Fas ligand. Cytokine-treated islets expressing IRS2(5A) secreted significantly more insulin in response to glucose than did islets expressing IRS-2(WT). This could be attributed to the higher transcription of Pdx1 in cytokine-treated islets that expressed IRS-2(5A). Accordingly, transplantation of 200 islets expressing IRS2(5A) into STZ-induced diabetic mice restored their ability to respond to a glucose load similar to naïve mice. In contrast, mice transplanted with islets expressing IRS2(WT) maintained sustained hyperglycemia 3 days after transplantation.

Conclusions: Elimination of a physiological negative feedback control mechanism along the insulin-signaling pathway that involves Ser/Thr phosphorylation of IRS-2 affords protection against the adverse effects of proinflammatory cytokines and improves beta-cell function under stress. Genetic approaches that promote IRS2(5A) expression in pancreatic beta-cells, therefore, could be considered a rational treatment against beta-cell failure after islet transplantation.

Figure 7: Effects of transplantation of islets overexpressing IRS-25A on blood glucose levels of diabetic mice. A: Two hundred islets infected with an empty vector control were transplanted under the left renal capsule of STZ-treated hyperglycemic syngeneic mouse. Immunohistochemistry, carried out 20 days after transplantation of the grafted islets, reveals an overlap between GFP-expressing cells and insulin positive cells (C-renal capsule, G-grafted islets, K-kidney). B: Two hundred islets, infected with empty/control adenoviral constructs, IRS-2WT, or IRS-25A were transplanted under the kidney capsule of STZ-treated diabetic syngeneic mice (n = 6). Noninfected (healthy) and STZ-treated (diabetic) animals served as control. Blood glucose levels of overnight fasted mice. ***P < 0.001 between glucose levels of both IRS-2WT and IRS-25A and diabetic mice. C: Three days after transplantation, mice were fasted for 16 h and glucose tolerance test was performed. Data are means ± SEM of the indicated number of mice per group. *P <0.05 between glucose levels of mice transplanted with islets expressing IRS-25A and IRS-2WT. (A high-quality color representation of this figure is available in the online issue.)

Mentions:
To determine whether ex vivo introduction of IRS-25A into pancreatic islets prior to their transplantation improves the functionality of the transplanted β-cells, C57BL/6J mice were rendered diabetic by a single high-dose injection of STZ. Mice were transplanted five days later when their fasting blood glucose levels exceeded 400 mg/dl. Islets used for transplantation were isolated from healthy syngeneic mice and were infected with adenoviruses harboring either IRS2WT, IRS25A, or empty GFP control. As shown in Fig. 7A, the transplanted islets remained as a packed mass under the kidney capsule for 20 days after transplantation. Most transplanted cells retained their insulin content (Fig. 7A, middle) and expressed the adenoviral constructs, revealed by their GFP staining (Fig. 7A, left). As expected, the grafted islets exhibited an overlap between GFP expression and insulin-positive cells (Fig. 7A, right).

Figure 7: Effects of transplantation of islets overexpressing IRS-25A on blood glucose levels of diabetic mice. A: Two hundred islets infected with an empty vector control were transplanted under the left renal capsule of STZ-treated hyperglycemic syngeneic mouse. Immunohistochemistry, carried out 20 days after transplantation of the grafted islets, reveals an overlap between GFP-expressing cells and insulin positive cells (C-renal capsule, G-grafted islets, K-kidney). B: Two hundred islets, infected with empty/control adenoviral constructs, IRS-2WT, or IRS-25A were transplanted under the kidney capsule of STZ-treated diabetic syngeneic mice (n = 6). Noninfected (healthy) and STZ-treated (diabetic) animals served as control. Blood glucose levels of overnight fasted mice. ***P < 0.001 between glucose levels of both IRS-2WT and IRS-25A and diabetic mice. C: Three days after transplantation, mice were fasted for 16 h and glucose tolerance test was performed. Data are means ± SEM of the indicated number of mice per group. *P <0.05 between glucose levels of mice transplanted with islets expressing IRS-25A and IRS-2WT. (A high-quality color representation of this figure is available in the online issue.)

Mentions:
To determine whether ex vivo introduction of IRS-25A into pancreatic islets prior to their transplantation improves the functionality of the transplanted β-cells, C57BL/6J mice were rendered diabetic by a single high-dose injection of STZ. Mice were transplanted five days later when their fasting blood glucose levels exceeded 400 mg/dl. Islets used for transplantation were isolated from healthy syngeneic mice and were infected with adenoviruses harboring either IRS2WT, IRS25A, or empty GFP control. As shown in Fig. 7A, the transplanted islets remained as a packed mass under the kidney capsule for 20 days after transplantation. Most transplanted cells retained their insulin content (Fig. 7A, middle) and expressed the adenoviral constructs, revealed by their GFP staining (Fig. 7A, left). As expected, the grafted islets exhibited an overlap between GFP expression and insulin-positive cells (Fig. 7A, right).

Bottom Line:
Cytokine-treated islets expressing IRS2(5A) secreted significantly more insulin in response to glucose than did islets expressing IRS-2(WT).Elimination of a physiological negative feedback control mechanism along the insulin-signaling pathway that involves Ser/Thr phosphorylation of IRS-2 affords protection against the adverse effects of proinflammatory cytokines and improves beta-cell function under stress.Genetic approaches that promote IRS2(5A) expression in pancreatic beta-cells, therefore, could be considered a rational treatment against beta-cell failure after islet transplantation.

Objective: Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-1 signaling. Here, we examined the role of Ser phosphorylation of IRS-2 in mediating the inhibitory effects of proinflammatory cytokines and cellular stress on beta-cell function.

Research design and methods: Five potential inhibitory Ser sites located proximally to the P-Tyr binding domain of IRS-2 were mutated to Ala. These IRS-2 mutants, denoted IRS-2(5A), and their wild-type controls (IRS-2(WT)) were introduced into adenoviral constructs that were infected into Min6 cells or into cultured murine islets.

Results: When expressed in cultured mouse islets, IRS-2(5A) was better than IRS-2(WT) in protecting beta-cells from apoptosis induced by a combination of IL-1beta, IFN-gamma, TNF-alpha, and Fas ligand. Cytokine-treated islets expressing IRS2(5A) secreted significantly more insulin in response to glucose than did islets expressing IRS-2(WT). This could be attributed to the higher transcription of Pdx1 in cytokine-treated islets that expressed IRS-2(5A). Accordingly, transplantation of 200 islets expressing IRS2(5A) into STZ-induced diabetic mice restored their ability to respond to a glucose load similar to naïve mice. In contrast, mice transplanted with islets expressing IRS2(WT) maintained sustained hyperglycemia 3 days after transplantation.

Conclusions: Elimination of a physiological negative feedback control mechanism along the insulin-signaling pathway that involves Ser/Thr phosphorylation of IRS-2 affords protection against the adverse effects of proinflammatory cytokines and improves beta-cell function under stress. Genetic approaches that promote IRS2(5A) expression in pancreatic beta-cells, therefore, could be considered a rational treatment against beta-cell failure after islet transplantation.